The present invention relates to a substrate suitable for use with surface plasmon resonance (SPR) imaging and other SPR techniques, and in particular to a versatile substrate providing improved sensitivity and ease of use in the identification, detection and quantification of DNA, RNA, proteins, and other biomolecules.
Referring to FIG. 1 in SPR imaging, a polarized light beam 10 may be directed to the rear side of a transparent substrate 12 at an angle θ as coupled to the transparent substrate 12 by prism 14. The light beam 10 passes through the substrate 12 to reflect off a rear surface of a metallic film 18 adhered to a front surface 20 of the substrate 12. In imaging SPR, the light is received by a camera and the image produced by the camera is analyzed.
The front and exposed surface of the metallic film 18 may have different probe molecules 22 attached to it. These probe molecules 22 may be exposed to a carrier stream 24 containing target molecules 26 which may selectively react with ones of the probe molecules 22 according to a designed experiment.
The intensity of reflected light beam 10′ from different points on the rear surface of the metallic film 18 will be dependent on the density of material (probe molecules 22 and target molecules 26) attached to the front side of the metallic film 18. This variation is caused by a change in reflectivity (% R) of the metallic film 18 caused by modification of surface plasmon resonance of the metallic film 18 by the material on the front side of the metallic film.
Referring to FIG. 2 for a given angle θ1, the reflectivity of the rear of the metallic film 18 at any given point will vary from value R1 to value R2 as the reflectivity curve 27 shifts rightward to reflectivity curve 27′ with the increase in material attached to the front side of the metallic film 18. A precise measurement of reflectivity R can thus reveal a location of the binding of the target molecules 26. This location, together with the known location of complementary probe molecules 22, can reveal the type of target molecules 26 in the carrier.
SPR imaging normally looks at a difference in reflectivity (R1) of the substrate prior to reaction with the target molecules 26 and in reflectivity (R2) of the substrate after reaction with the target molecules to accentuate the changes caused by the binding of probe molecules 22 and target molecules 26 to reduce baseline variations in reflectivity caused by the probe molecules 22, variations in the metallic film 18 and substrate 12, and changes in the material of the front surface metallic film 18.
SPR imaging delivers sensitive detection of target molecules without the need to label the target molecules, for example, with fluorescent or radioactive materials as is otherwise necessary to detect concentrations of target molecules at particular locations.